Cloning and Expression of Soluble Cytochrome C and Its Role in Polyvinyl Alcohol Degradation by Polyvinyl Alcohol-Utilizing Sphingopyxis Sp. Strain 113P3

J Biosci Bioeng. 2008 Feb;105(2):147-51. doi: 10.1263/jbb.105.147.

Abstract

The gene encoding cytocrome c in the pva operon of Sphingopyxis sp. strain 113P3 was cloned, on the basis of the sequence of the gene for cytochrome c (GenBank accession no. AB190288). The deduced amino acid sequence of the gene showed homologies (37% and 47% identities) with two cytochromes c of different origins. The recombinant cytochrome c tagged with hexahistidines was expressed in the periplasm of Escherichia coli BL21(DE3) harboring pT-GroE, which was in accordance with the localization of cytochrome c in strain 113P3; the protein was purified to homogeneity. The purified recombinant cytochrome c was a monomeric protein with a molecular weight of 16.5 kDa. The oxidized and reduced forms of the protein showed absorption maxima at 409 nm and at 414, 520 and 550 nm, respectively. The recombinant cytochrome c was fully reduced by polyvinyl alcohol (PVA), coupled with a catalytic amount (1/10 molar concentration) of the recombinant PVA dehydrogenase (PVADH) of the same origin, suggesting that the cytochrome c involved in the pva operon is a physiological primary electron acceptor for PVADH and that PVA dehydrogenation is linked with the respiratory chain in Sphingopyxis sp. strain 113P3.

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Alphaproteobacteria / genetics
  • Alphaproteobacteria / metabolism*
  • Cloning, Molecular / methods*
  • Cytochromes c / chemistry*
  • Cytochromes c / genetics
  • Cytochromes c / metabolism*
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Enzymologic / physiology
  • Polyvinyl Alcohol / chemistry*
  • Polyvinyl Alcohol / metabolism*
  • Recombinant Proteins / metabolism
  • Solubility

Substances

  • Recombinant Proteins
  • Polyvinyl Alcohol
  • Cytochromes c
  • Alcohol Oxidoreductases
  • polyvinyl-alcohol dehydrogenase (acceptor)